Induction of endothelin-1 expression by oxidative stress in vascular smooth muscle cells.

Atherosclerosis is based on endothelial dysfunction leading to impaired vasomotor function. This is partially due to nitric oxide (NO) depletion caused by oxidative stress. Since the vasoconstrictor endothelin-1 (ET-1) might also be involved in endothelial dysfunction, we investigated whether oxidative stress regulates ET-1 expression in vascular smooth muscle cells (VSMC). Human aortic VSMC were treated with H(2)O(2) (200 microM) for up to 8 h. mRNA expression of preproendothelin (prepro-ET) was analyzed by RT-PCR. ET-1 protein and the marker for oxidative stress, 8-isoprostane, were determined by ELISA. Activity of cytosolic phospholipase A2 (cPLA(2)) as an indicator of ET-1 autocrine activity was measured photometrically. Stimulation of VSMC with H(2)O(2) resulted in increased expression of prepro-ET mRNA after 1 h with a maximum after 6 h (fourfold), similar to treatment with angiotensin II. ET-1 protein was significantly increased by H(2)O(2) treatment with a maximum after 8 h (P<.05). This effect was inhibited by the antioxidants resveratrol (100 microM) and quercetin (50 microM). In quiesced VSMC, incubation with H(2)O(2)-conditioned medium resulted in increased cPLA(2) activity compared to the controls (P<.05). This activity was partially inhibited by the ET(A)-receptor antagonist, PD 142893 (10 microM), indicating functional ET-1 in the conditioned medium. The presence of oxidative stress in H(2)O(2)-treated VSMC was associated by significantly increased formation of 8-isoprostane (P<.05). The data indicate for the first time that oxidative stress increases ET-1 generation and autocrine ET-1 activity in VSMC, a mechanism that might contribute to endothelial dysfunction in atherosclerosis.